The present invention relates to methods and apparatuses for testing analysis fluids, and more particularly to a microcuvette for analyzing one or more components of a fluid. Significant contemplated applications of the invention are in the biological sciences, especially diagnostic medicine. In this field, analysis fluids would primarily be bodily fluids, notably whole blood.
Several dry chemistry technologies have been introduced in recent years for testing of blood specimens at the patient point-of-care (POC). Testing at the POC offers advantages of fast turnaround time, timely intervention, miniaturized and cost effective equipment, and improved patient outcomes. xe2x80x9cDry chemistryxe2x80x9d means that the chemical reagents are contained within a test strip device solely in dry, but not in liquid form. Since the reagents are more stable when stored in dry form, products employing dry reagent technology usually have longer shelf life than those using liquid reagents.
In most devices, the reagents are applied to the test strip by some impregnation or coating method whereby a liquid reagent is impregnated or coated onto an integrated reagent-carrying member. The reagent member can be a bibulous material (paper), a membrane, or a reagent film. After evaporation of the reagent solvent, the dry and stable reagent is then contained within a reactive zone, signal member test field of the device. As analysis fluid makes contact with the dry reagents, the reagents are generally at least partially re-solubilized so as to react with the analyte of interest.
The most substantial application of dry technology today is in the field of self-monitoring of blood glucose (SMBG) by millions of diabetics. In this field, both photometric and sensimetric detection technologies are applied for signal quantification. A large portion of metering systems currently used by practitioners employ reflectance photometry. In these meters, light integrating a wavelength absorbed by the colored reaction product of glucose is shined onto the surface of the test field. The test field is preferably mounted on a solid state backing, usually a white plastic material. In this fashion, no light can be transmitted, so that the unabsorbed, scattered portion of the light is reflected.
In contrast to conventional photometry where absorbance of a colored or UV-absorbing reaction product is measured from reduced light transmittance in the direction of the incident light beam, reflectance is typically measured at a location angled away from the direction of incident light. As light of varying incident wavelengths is reflected in different directions, an informed choice must be made as to which ranges of incident and reflective angles to select for obtaining a signal that is most sensitively and most specifically related to concentration. Preferably, the photocurrent detector (photodiode) of the metering device is positioned at a location where unspecific scattering is at a minimum and specific reflectance is at a maximum. However, since specific and unspecific reflectance can usually not be completely spatially separated, pure signals cannot be obtained. For these reasons, measurements made in the reflectance mode do not follow Lambert Beer""s law and are therefore fundamentally non-linear. This is in contrast to measurements made in the transmittance mode, which show linear signal-to-concentration responses of absorbance measurements.
Several more recent SMBG devices employ electro-sensimetric detection. The reaction current, measured by a miniature enzyme electrode, is related to glucose concentration and can be monitored amperometrically or by some other means of electrochemical detection. Most reflectance photometric and sensimetric systems employ in the first reaction step the oxygen-dependent enzymic oxidation of glucose by glucose oxidase. This reaction is specific for glucose and produces hydrogen peroxide as a reaction by-product from water and molecular oxygen. Some other systems use glucose dehydrogenase in conjunction with one or more electron acceptors.
In the reflectance photometric systems employing glucose oxidase, the generated hydrogen peroxide is reacted with peroxidase and a chromogen. The oxidized chromophore is then reflectance photometrically quantified by comparison to an on-board standard curve that relates reflectance signal to concentration. Quantification by nonlinear reflectance rather than linear absorbance photometry based on Lambert Beer""s law is necessary because the law only holds for clear, non-scattering layers.
Numerous clinical evaluations of currently used glucose metering devices have generally demonstrated adequate analytical performance. However, compromised performance on some of the products, and even outright erroneous results have also been reported. Manufacturers are therefore continually striving to minimize the technical complexity of the systems, maximize operational ease, and improve reliability. Because of the vast global dimension of the SMBG market and the fast growth of diabetes in the world, these efforts have huge socioeconomic implications. At current retail prices of test strips for SMBG, a compliant insulin-dependent diabetic spends in excess of $1000 annually on test strips only, constituting a total global test strip market in excess of $2.4 billion. While this cost can generally be absorbed by citizens or reimbursement systems of the western world, it is prohibitive for most people living in countries other than the western world, where the growth of diabetes is most rampant.
Depending on measurement principle, current test systems have their intrinsic advantages and limitations. An advantage of the reflectance photometric systems is that they measure color. Potentially, this enables both visual and instrumented signal recognition. Visual interpretation can serve as a confidence check for quantitative results provided by the meter. And in markets where meters are not readily available, concentration can still be determined semi-quantitatively. Visual recognition is still well accepted as it was the only method available when SMBG started on a larger scale in the late 1970""s. (A significant portion of the world market for glucose test strips is still visual at this time).
Unfortunately, the important feature of visual backup is realized only in a minority of currently marketed systems. This limitation resides in the method by which cellular component of blood is separated from plasma component. In older products, plasma was separated by soak through methods into coated bibulous materials or reagent films. Cells were then manually removed from the site of blood application by either washing or wiping them away, potentially giving rise to significant operator-induced errors. Several newer methods permit separation by means other than washing or wiping. The most frequently used are separation by porous glass fiber fleeces or membranes. In these matrices pore sizes are chosen so that cellular component is held back on the matrix surface, whereas plasma component diffuses through the separating member and into the detection member. Membranes are preferred as plasma separating materials over glass fiber fleeces because they generally absorb less blood. However, one notorious limitation of membranes is that the blood cells can clog pores. More recently, this problem has been largely overcome by using asymmetrical membranes in which pores have larger diameters on the side chosen for blood application as compared to the side dedicated to plasma retrieval.
In most current colorimetric test strips, the separating member is sandwiched against the detection member to provide for ready transfer of plasma into the reagent-impregnated detection member. The reflectance measurement is then made on the side of the test strip opposite to the side of blood application. To keep needed blood volume low, the thickness of the separation member is kept at a minimum. An adverse consequence is that the spatial separation of red cells from the site of measurement is then so small that the cells are incompletely shielded by the thin zone of separation material that is devoid of cells. In instrumented measurements, this xe2x80x9cshining throughxe2x80x9d effect of red cells can, as long as the effect is constant for each measurement, be corrected by calibration or a dual wavelength measurement. However, such corrective methodology makes measurements more complex and less precise. Another corrective method would be to insert an additional, optically dense layer or a contrast material such as titanium dioxide between separation and detection members. But use of this method would further increase blood volume and hence invasiveness. The shining through effect of red cells is particularly disadvantageous for visual interpretation. It is mainly for this reason that most present-day colorimetric test strips cannot be read visually. For the user, the potential of a visual confidence check on digital readouts is thus unfortunately lost.
Another drawback of having a discrete separating member is the well known phenomenon of dependency of test results from the ratio of cellular/plasma component, i.e. the often variable hematocrit. Most current SMBG systems produce results that are inversely correlated with hematocrit. This is because at high hematocrits, red cells can block free diffusion of plasma and hence glucose into the detection member, causing test results to be erroneously low.
Exemplary test strip devices of the present invention all but eliminate hematocrit dependence. (See, FIG. 11). It is hypothesized that absence of significant dependence is the result of supplying the blood to reagent films in a mobile fashion and over a specified period of time, wherein cells are continually removed by capillary force as the blood moves downstream through the collection capillary.
Good progress towards miniaturization was achieved with the advent of the electrochemical sensor methods, not because they would be innately more sensitive (they are not), but because they can function on whole blood as the analysis fluid, thereby obviating the need for a plasma-consuming blood separating member. In some of these products, miniaturization is further aided by provision of capillary fill techniques. Despite these improvements, a major limitation of the sensor methods is that visual backup is completely lost. The user has no other means of accepting a test result than complete reliance on the digitally displayed concentration numbers. This places a very heavy burden on the manufacturer as even minor flaws in test strip architecture or signal conductivity could have disastrous consequences. Also, as is the case with the reflectance photometric methods, signal-to-concentration responses of the sensor methods are not linear, necessitating complex mathematical modeling for device calibration. A further limitation of sensimetric systems is that expansion of the test menu to include analytes other than glucose is quite impractical because a different enzyme electrode would be needed for each additional analyte. By contrast, using the method of the candidate device, test strips for additional analytes could easily be developed by simply substituting detection enzymes in the reagent film. Furthermore, hematocrit dependence in sensor methods can be substantial due to xe2x80x9cdilutionxe2x80x9d of the electrochemical reaction milieu by cellular component. Last not least, the technical sophistication and ensuing manufacturing complexity of the sensor methods makes it much more challenging to manufacture them at low cost.
Measured by its unique performance assets of removal of particulate matter by a capillary force gradient, nano-volume miniaturization, the capacity for transmittance measurement of colorimetric signals, and architectural and manufacturing simplicity, the applicant clear film technology stands on an elevated technological platform for which there is essentially no directly competing prior art to cite.
Previous attempts at technically achieving the desired criteria of testing simplification and miniaturization, so that test results could be easily obtained by practitioners at the POC and even patients in their homes, can conceivably be divided into several enabling technology categories of whole blood separation/plasma retrieval. These are separation by: A) glass fiber matrices (fleeces, xe2x80x9cpapersxe2x80x9d) only; B) membranes only; C) combined arrangements of glass fiber (pure or composite) and membrane matrices; D) separations facilitated by agglutinating agents (e.g. lectins, red cell antibodies, carbohydrates, amino acids, etc.); E) separation by soak-in methods into polymers or xe2x80x9cgelsxe2x80x9d (e.g. wiping or washing of cells); F) separation or whole blood delivery augmented by capillary elements.
A method that in some of its principles resembles the applicant technology (and provided significant intellectual fuel for its discovery and development) was disclosed by Azhar et al. (U.S. Pat. No. 5,260,195). However, the core subject of this patent is the description of a manufacturing process from water-insoluble monomers for an acrylic copolymer (latex) that displays the desired properties of reagent film rehydration and filter-less plasma retrieval. Because of the relative water insolubility of the monomers and the copolymer formed from them, this process must rely on the use of organic solvents. Use of organic solvents during manufacturing is undesirable, because of associated environmental, health and cost considerations. The authors of this patent also show a picture of an xe2x80x9capparatusxe2x80x9d (a test strip) apparently using the described copolymer. This apparatus is also made subject to one of the claims. A rectangular capillary space is created in the apparatus by lamination of continuous plastic template strips so that the capillary space extends over the entire dimensions of the reagent film. Unfortunately, description of the apparatus and how it might be manufactured is extremely limited. Brief mention is made of the method of blood removal from the capillary vessel. This is to be performed manually by pressing a cotton swab against one of the two open sides of the capillary and waiting for all sampled blood to be absorbed by the swab. While the method appears to work in principle, it is obviously inflicted with all of the known limitations of manual handling of blood specimens, e.g. dosing imprecision, incomplete reagent and sample mixing, variations in reaction timing, and potential danger of infection.
Accordingly, there is a need for a test strip device that: 1) does not require integration of a blood spreading or plasma separating member within the architecture of the test strip device, i.e. can be performed on less than one (1) microliter of blood; 2) can accommodate all types of photometry as detection principles; 3) streamlines calibration procedures owing to linear signal-to-mass responses of absorbance measurements; 4) is simple by design and thus operationally rugged and analytically precise; 5) features visual backup for users using meters, and visual semi-quantification for users not using meters; 6) can be performed in less than 30 sec by non-technical personnel; 7) can be mass manufactured easily and cost-effectively.
The exemplary test strip devices disclosed herein are believed to be the first combining colorimetric detection with capillary fill sampling and emptying, and the first measuring transmittance by virtue of using a clear polymer as reagent film, wherein the polymer is dispersed on translucent plastic support. Owing to the partial water permeability of the reagent film, components of aqueous analysis fluid can enter the reagent film upon rehydration of the film by analysis fluid. This process of rehydration of the reagent film enables reaction of a component of the analysis fluid, i.e. the analyte, with the reagent encapsulated in the film.
Pursuant to a first embodiment, there is provided a miniaturized capillary channel test strip device manufactured by plastic flow injection molding. The capillary channel doses and transports analysis fluid, e.g. whole blood within open capillary space extending through the plastic casing of the device. The plastic casing defines the interior capillary dimensions and also serves as a protective housing for a reagent film dispersed within the device The capillary channel includes a collection component and a wicking component, wherein the movement of analysis fluid from sampling site to collection site to wicking site is effected by a gradient of capillary force. The gradient is induced by specialized designs in which the surface/volume ratio of the wicking component is in excess of the surface/volume ratio of the collection component. This differential in surface/volume ratio of wicking component and collection component induces a capillary gradient acting in the downstream direction. Advantageously, the differential in capillary force becomes the main driver for fluid transport through the channel.
The wicking component can either be an absorptive material (e.g. sponge), or it can itself be a capillary or a system of capillaries composed of a plurality of individual wicking capillaries.
The collection component incorporates a planar reagent film (test field). The reagent film contains dried chemical reagents capable of reacting with an analyte, and an inert dried polymer capable of spontaneous rehydration, to thereby absorb a defined portion of an analysis fluid. A unique feature of the invention is that the analysis fluid can be either a homogeneous solution, or a non-homogeneous mixture containing cellular or other particulate matter suspended in the fluid. When the analysis fluid is whole blood, the polymer absorbs a defined volume of blood plasma while inhibiting cellular component of blood from penetrating the reagent film surface. The cellular component is wholly removed from the reagent film by the gradient of capillary force, thereby obviating the need for a separate cell filtering material, and unmasking the reacted test field for visual or instrumented analysis. The instrumented analysis can be performed by fluorimetry, luminescence, reflectance, and preferably by transmittance photometry performed on translucent reagent films.